This grant supports all research activities in structural enzymology carried out by the Stoddard lab. Three interconnected projects are covered under this grant: 1. Structure determination and functional studies of novel biological catalysts, primarily endonucleases and splicing factors encoded within mobile introns. In the previous funding period, the structure of the I-CreI and I-PpoI homing endonucleases were determined, both free and bound to DNA. Additional studies describing the catalytic mechanism and thermodynamics of site recognition and binding for I-PpoI were also reported. Dr. Stoddard proposes to extend his studies on these two systems in the next funding period, and to determine the structures of several new proteins from related enzyme families: I-AniI, I-NanI, I-SspI and I-Tevill. 2. Continued development of time-resolved crystallographic methods to determine the structures of catalytic intermediates, and to study reaction profiles based on these structures. In the previous funding period, studies using photolytic triggering, Laue diffraction, mutagenesis, and solvent exchange were reported using several model systems. Dr. Stoddard proposes to continue these studies using three model systems: I-PpoI, the hammerhead ribozyme and ornithine aminotransferase. Dr. Stoddard also proposes to develop methods to model the reaction coordinates and free energy profiles of individual catalytic reaction steps, using structures of discrete intermediates as well-defined points that define the ends of the pathway. 3. Exploitation of allosteric regulatory sites as targets for structure-based drug design. In the previous funding period, structural studies of the allosteric regulation of pyruvate kinase were reported, followed by biochemical, genetic and computational studies that demonstrate the effect of PK deregulation on cell cycle progression and that identified novel, synthetic allosteric effectors of the enzyme. Dr. Stoddard proposes to continue these studies by performing similar experiments on the human R and M2 isozymes of PK, and on human ribonucleotide reductase.